Abuse of cocaine is an intractable social and medical problem that is resistant to remediation through pharmacotherapy. Cocaine acts to block the reuptake of monoamines, dopamine, norepinephrine, and serotin thus prolonging and magnifying the effects of these neurotransmitters in the central nervous system (Benowitz N L (1993) Pharmacol Toxicol 72, 3-12). Cocaine toxicity is marked by both convulsions and cardiac dysfunction (e.g., myocardial infarction, cardiac arrhythmias, increased blood pressure, stroke, or dissecting aneurysm, and increased myocardial oxygen demand), due to effects on neurotransmitter systems and myocardial sodium channel blockade (Bauman J L and DiDomenico R J (2002) J Cardiovasc Pharmacol Ther 7, 195-202; Wilson L D and Shelat C (2003) J Toxicol Clin Toxicol 41, 777-788; Knuepfer M M (2003) Pharmacol Ther 97, 181-222). Because of cocaine's ability to readily cross the blood brain barrier and its widespread effects on the central and peripheral nervous systems, overdose can result in sudden death (see Bauman J L and DiDomenico R J (2002) J Cardiovasc Pharmacol Ther 7, 195-202, for review).
Although the mechanism of cocaine's action is well understood, this information has not yet resulted in the development of an effective antagonist of cocaine that could be used in abuse and overdose situations. The rapid and pleiotropic effects of cocaine present a complex problem for the treatment of acute cocaine toxicity (Carroll F I, Howell L L and Kuhar M. J (1999) J Med Chem 42, 2721-2736). The two types of therapies that are available for the treatment of opioid abuse, antagonism (e.g., naltrexone) and replacement (e.g., methadone), do not have parallels in the case of cocaine, although attempts at the latter are being considered (e.g., J. Grabowski et al. (2004) Addictive Behaviors 29, 1439-1464). One approach is to prevent or reduce the cocaine from reaching sites of action by administering either endogenous esterases, cocaine specific antibodies, or a catalytic antibody.
Naturally occurring cocaine is hydrolyzed at the benzoyl ester by serum butyrylcholinesterase (BChE) to nontoxic ecgonine methyl ester and benzoic acid. In the liver, carboxylesterase hCE-2 hydrolyzes the methyl ester to yield benzoylecgonine and methanol (see e.g., FIG. 1). The elimination half-life of cocaine in the blood ranges from 0.5 to 1.5 hr (T. Inaba (1989) Canadian Journal of Physiology & Pharmacology 67, 1154-1157). There have been a few attempts to use naturally occurring BChE or genetically engineered BChE to increase cocaine breakdown (see e.g., Carmona et al. (2000) Drug Metabolism & Disposition 28, 367-371; Xie et al. (1999) Molecular Pharmacology 55, 83-91; Sun et al. (2002a) Molecular Pharmacology; Sun et al. (2002b) Pharmacology & Experimental Therapeutics 302, 710-716; Duysen et al. (2002) Journal of Pharmacology & Experimental Therapeutics 302, 751-758; Gao Y and Brimijoin S (2004) Journal of Pharmacology & Experimental Therapeutics 310, 1046-1052; Gao et al. (2005) Molecular Pharmacology 67, 204-211). Other researchers have utilized a monoclonal antibody, Mab 15A10, as a catalytic antibody to cocaine (see e.g., Landry et al, 1993; Mets et al., 1998; Baird et al., 2000; Larsen et al., 2004), while others are exploring the use of cocaine vaccines (see e.g., Kosten et al. (2002) Vaccine 20, 1196-1204).
TABLE 1Kinetics of several cocaine hydrolyzing enzymes against (-) cocaine.KcatKmEfficiencyEnzyme(min − 1)(μM)(kcat/Km)ReferenceBChE4.14.59.1 × 106Sun et al., 2002aAla328W/Y332A154188.5 × 106Sun et al., 2002aMab15A102.2220  1 × 104Larsen et al., 2004AME 359620203.1 × 107Gao et al., 2005CocE4680.647.2 × 108Turner et al., 2002
A bacterium, Rhodococcus sp. MB 1, indigenous to the soil surrounding the coca plant, has evolved the capacity to utilize cocaine as its sole carbon and nitrogen source. The bacterium expresses a cocaine esterase (CocE) that acts similarly to BChE to hydrolyze the benzoyl ester of cocaine, yielding ecgonine methyl ester and benzoic acid (see e.g., FIG. 1) (Bresler et al. (2000) Appl Environ Microbiol 66, 904-908; Turner et at. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Struct Biol 9, 17-21). The gene for CocE has been isolated and cloned (Bresler et al. (2000) Appl Environ Microbiol 66, 904-908), and the crystal structure of CocE has been determined (Turner et at. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Struct Biol 9, 17-21). The structure of CocE (see e.g., FIG. 2) reveals a classic serine esterase fold in addition to two other domains that combine to form a cocaine binding pocket. Altering any of three amino acids (Asp, His, or Ser) within the catalytic triad in the active site (for review, see Dodson G and Wlodawer A (1998) Trends Biochem Sci 23, 347-352) inactivates the esterase activity against cocaine. Furthermore, mutation of residues that make contact with the benzoate moiety of cocaine (e.g., Tyr44) also disrupts cocaine hydrolysis, presumably through impairing oxyanion stabilization in the transition state (Turner et al. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Structural Biology 9, 17-21). The purified enzyme (MW ˜65 kDa) catalyzes cocaine very efficiently with Michaelis-Menten kinetics kcal=7.2 s−1 and Km=640 nM (Turner et al. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Structural Biology 9, 17-21), nearly three orders of magnitude greater than endogenous esterases and, most likely, would act quickly enough to detoxify humans who have overdosed on cocaine (Landry et al. (1993) Science 259, 1899-1901; Mets et al. (1998) National Academy of Sciences of the United States of America 95, 10176-10181). Additionally, the esterase also metabolizes cocaethylene, a potent metabolite of cocaine and alcohol, almost as efficiently as it metabolizes cocaine (kcat=9.4 s−1 and Km=1600 nM) (Turner et al. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Structural Biology 9, 17-21).
Thus, it would be desirable to provide a stable CocE for anti-cocaine therapeutics.